Liposomal recombinant human superoxide-dismutase for the treatment of peyronie&#39;s disease

ABSTRACT

Superoxide dismutase (SOD), preferably rhSOD, is used in liposomes, optionally mixed with hyaluronic acid and/or at least one physiologically acceptable carrier, and other optional additives, to prepare a pharmaceutical composition useful against increased concentrations of superoxide radicals and/or the damage caused thereby. The compositions are administered topically, preferably as gels or ointments, to prevent and/or heal human fibrotic tissue degeneration diseases, in particular induratio penis plastica (Peyronie&#39;s Disease).

This application is a continuation-in-part of application Ser. No.08/836,185, filed May 5, 1997, now U.S. Pat. No. 5,942,245, which inturn is a U.S. national stage application under 35 U.S.C. §371 ofPCT/EP95/04352, filed Nov. 6, 1995. The entire disclosures of both ofthese applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of superoxide dismutase (SOD),preferably of recombinant human SOD, in liposomes, for the preparationof pharmaceutical compositions, optionally mixed with hyaluronic acidand/or at least one physiologically acceptable carrier and/or otheroptional additives, for therapeutic and/or prophylactic use againstincreased concentration of superoxide radicals and/or damage causedthereby. More in particular, the present invention relates to the use ofhuman SOD packed within liposomes in the treatment of human fibrotictissue degeneration diseases, including induratio penis plastica (IPP),also commonly known as Peyronie's Disease.

2. Discussion of Related Art

Superoxide radicals are extremely reactive intermediate forms of thenatural oxygen molecule and, as a result of this property, canirreversibly damage organic compounds in the cells of the human body. Asprotection from the dangerous effect of these superoxide radicals, thecells have an enzyme which is capable of rapidly converting suchsuperoxide radicals into the more rapidly metabolizable and less toxichydrogen peroxide (H₂O₂).

Thereafter, the hydrogen peroxide, which is still toxic, is usuallydecomposed by the enzyme catalese into the harmless components water andoxygen.

The enzyme superoxide dismutase (SOD) occurs both in the human andanimal body and in plants and presumably in all microorganisms whichcome directly into contact with atmospheric oxygen (aerobic bacteria andfungi). In the cells of higher organisms (eucaryotes), there are mainlytwo types of this SOD: a manganece-containing SOD which is localized inthe mitochondria and is very similar to the bacterial SOD, and a secondone which is present freely in the cytosol and contains copper and zincatoms.

Unless stated otherwise, the term SOD is to be understood below asmeaning mainly Cu,Zn-SOD except for that from bovine blood erthrocytes,and bacterial or mitochondrial Mn-SOD and/or Fe-SOD, and recombinanthuman Cu,Zn-SOD (rhSOD).

Superoxide dismutase has been known under the name Orgotein since 1939,but the dismatuse activity was not discovered and described until 1969,by McCord and Fridovich. Its practical use was limited in the past inparticular by the short life or short biological availability of theprotein under natural conditions, which of course has an adverse effecton the frequency of the dosage intervals, the therapeutic doses to bechosen and the associated costs.

The most investigated and used SOD to date was the Cu,Zn-SOD from bovineblood erythrocytes. Following severe and in some cases even fataladverse reactions in the clinical-therapeutic use of bovine blood SOD,especially against arthritic diseases, preparations containing bovineblood SOD were prohibited, for example, in Austria. The preparation ofrecombinant human SOD, as described, for example, in AT 397 812 (PolymunScientific, 1994), offers a way out of this situation.

Inter alia, the incorporation of SOD molecules in liposomes is apossible method for controlling the short life or short biologicalavailability of the active substance SOD (Senga et al. 1990, Transplant.Proc. 22:2025).

The very first trial applications related to the treatment ofinflammations and inflammatory processes of the skin. However, uses inosteoarthritis and rheumatic arthritis are now also reported in theliterature (Hartmann et al. 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7142;Bannister et al. 1987, Critical Rev. Biochem 22:111). Especially in thearea of medicine, it is also reported that SOD improves the storabilityof organs for the purpose of a subsequent transplantation (Olson et al.1988, Transplant. Proc. 20:961), and a use for food preservation hasalready been mentioned (WO 85/01503).

SUMMARY OF THE INVENTION

The primary object of the present invention is to provide pharmaceuticalcompositions for transporting SOD, preferably rhSOD enclosed inprotective liposomes, in a gentle, effective manner and with betterbioavailability, to those parts of the body which are to be treated. Inspite of a continuing general prejudice on the part of those skilled inthe art, the inventors have been able in particular to use liposomallypackaged SOD successfully for human fibrotic tissue degenerationdiseases such as Peyronie's Disease, for bums and scalds and forradiation damage, caused, for example, by UV radiation or ionizingradiation, in particular by external application. In the case ofexposure to radiation, prophylactic use, for example together with aradiation-filtering or radiation-absorbing screening agent, is alsopossible in addition to therapeutic use.

A further object is to provide a pharmaceutical composition based on SODin liposomes, which overcomes the stated disadvantages of the prior artand thus opens up additional fields of use, in particular in the area ofcosmetics.

It is a further object of the present invention to transport SOD,preferably rhSOD enclosed in protective liposomes, in a gentle,effective manner and with better stability and a longer lasting effect,to the organic materials to be treated, for example vegetable or animaltissues, organs, organ or tissue transplants, cosmetic preparationsbased on organic substances and/or foods.

A particular object of the present invention is use of the synergisticeffect of a mixture of hyaluronic acid and SOD. Hyaluronic acid has alsorecently become known to those skilled in the art for its “free radicalacceptor properties”, and its use in wound treatment has repeatedly beendescribed (Amgen, WO 214480, 1992). The use of hyaluronic acid togetherwith Colony Stimulating Factor (CSF) or Platelet Derived Growth Factor(PDGF) for accelerating wound healing has also been described in theliterature (Zymogenetics, U.S. Pat. No. 5,128,321, 1992), and hyaluronicacid has also been described as an additive in cosmetics andpharmaceutical preparations (Shiseido, WO 9104279, 1991).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Surprisingly—a combination of hyaluronic acid and SOD, in particular amixture of hyaluronic acid and liposomally incorporated SOD, has notbeen described to date. To what extent this represents or represented aprejudice on the part of se skilled in the art cannot be adequatelyassessed at the present time. In any case, it was surprisingly found, inthe course of the experiments which have finally led to the presentinvention, that hyaluronic acid supports and reinforces the effect ofSOD, also of liposomally incorporated SOD, in an almost ideal manner.

In the course of the experiments which have finally led to the presentinvention, it was also surprisingly found that, thanks to the inclusionof the SOD molecules in liposomes, not only was their stability andbioavailability increased but also the concentration of SOD required toachieve the desired effect could be reduced by a factor of 10 comparedwith SOD compositions without liposomes, without having to accept loweractivity. Moreover, because of the liposomal dosage form, aphysiologically more advantageous dosage of the active substance at theplace of demand is also achieved, which of course has a veryadvantageous effect on the amount and/or frequency of the doses and theassociated costs. This advantageous effect of the physiologically moreadvantageous dosage of liposomally incorporated SOD on site also plays arole in inflammations and inflammatory processes at the body surface andin the interior of the body and in processes for improving the stabilityof organic materials.

It is however also presumed that the direct enzymatic action of the SODprotein may not be the only decisive factor for the success of healing.It is known that other enzymes, e.g., cytochrome A, histone, lysozymeand ribonuclease, for example in dimerized form, have additionalproperties which considerably extend those of the monomer, the actionspectrum and the respective field of use of the dimeric proteins thusbeing advantageously extended in comparison with the monomers(Bartholeyns and Zeneburgh, Europ. J. Cancer, Vol. 15, 1979, 85-91).

In the course of enclosing the SOD in the liposomes according to theinvention, as described, for example, in Example 1, at least a part ofthe SOD molecules having a molecular weight of about 32000 Daltonagglomerate to give aggregates of 2 or 3 molecules. Although it has notyet been demonstrated, it is possible that these aggregates result in anadditional advantageous effect, for example with regard to increasedstimulation of phagocytosis activities.

With the use of hyaluronic acid mixed with SOD, in particular rhSOD,with or without liposomes, it has surprisingly been found that asynergistic effect occurs in so far as, particularly in the case ofexternal application, substantially smoother and more elastic new tissueforms in comparison with an SOD-treated bum wound without hyaluronicacid. This effect was also found in the case of inflamed wounds on thebody surface. Hyaluronic acid mixed with liposomal SOD serves both as asynergistic free radical acceptor as a pharmaceutically acceptablecarrier.

In the experiments on which the present invention is based, it was alsounexpectedly found that with the use of a composition of hyaluronic acidmixed with SOD, in particular rhSOD in liposomes, a synergistic effectis obtained in so far as a significantly longer stability or duration ofaction of the composition itself in comparison with the SOD treatmentwithout hyaluronic acid, and hence also a longer stability of thetreated materials, result; this effect is evident in particular when theSOD is present freely in the composition, i.e., not enclosed inliposomes. Both SOD alone and hyaluronic acid alone were substantiallyinferior in this effect to a mixture of the two components.

This synergistic effect of a mixture of SOD and hyaluronic acid ispossibly promoted because the hyaluronic acid both protects thephospholipid layers of the liposomes and/or the sensitive SOD moleculesfrom harmful, i.e., mainly oxidizing, influences from outside and, afterdeactivation of the SOD, also makes its own, although only small,contribution to the elimination of superoxide radicals.

The present invention includes several different embodiments of the useof SOD, in particular rhSOD in liposomes, with or without hyaluronicacid and optionally in combination with carriers and/or furtheradditives, for the preparation of pharmaceutical compositions against anumber of indications.

One embodiment or one feature of the invention relates to the use ofSOD, in particular of recombinant human SOD (rhSOD) in liposomes,optionally mixed with hyaluronic acid and/or a physiological acceptablecarrier and optionally further additives usually used for pharmaceuticalformulations, for the preparation of pharmaceutical compositions againsthuman fibrotic tissue degeneration diseases, in particular Peyronie'sDisease.

Peyronie's disease presents a variety of symptoms due to structuralalterations of the penile corpora cavemosa: in a series of 100 patientswe found penile deviation in 77%, cavemosal plaques in 79%, pain at thesite of the lesion in 60% and erectile dysfunction in 23%. Thesesymptoms may interfere with the ability of having satisfactory sexualintercourse.

While surgery is only indicated in case of severe penile angulation,various non-surgical therapies have been applied during the last decadesto treat minor deformities or painful lesions. However, most of theseconservative regimens have considerable side effects including reddeningand swelling of the skin, allergic reactions of all degrees, and evenanaphylactic and anaphylactoid reactions.

Intralesional injections (e.g., using steroids, bovine SOD “orgoteine”,collagenase, verapamil, interferon alpha) are extremely painful andrequire local anesthesia. Para-aminobenzoate, the most popular oral drugfor Peyronie's disease therapy, provokes considerable gastro-intestinalsymptoms in most patients and, moreover, failed to prove efficacy in acontrolled study recently.

The optimal drug profile for Peyronie's disease treatment shouldtherefore include painless topical application, no adverse reactions andproven efficacy. An ointment or gel with an active ingredient thatpenetrates to the cavernosous lesion and effectively reduces Peyronie'ssymptoms would be an ideal therapy.

The present invention meets these needs and solves the problems with thetreatment of IPP by topically administering an effective dose of humanSOD, preferably human recombinant Cu,Zn-SOD, in liposomes to theconcerned lesion sites or fibrotic tissues.

The use of the liposomally packaged human SOD has the advantage of animproved pharmacokinetic profile including increasing transdermalpenetration of the composition containing the human SOD, protecting theactive ingredient SOD against immediate tissue reaction, and ensuring aslow SOD release at the site of desired action. These significantadvantages are further completed by the beneficial change in the mode ofadministration, in that thanks to the liposomal formulation of theSOD-drug in the form of a gel, a cream or an ointment, the patient needno longer painfully inject himself with a liquid medication but insteadmay simply externally administer the SOD preparation.

Bovine SOD (orgoteine) has been in use for the treatment of rheumaticdiseases and a variety of inflammatory disorders in the 1980's.Urologists injected SOD into Peyronie's plaques as well as into urinarybladders (in patients suffering from interstitial or radiationcystitis). Preliminary reports suggested a success rate in Peyronie'sdisease that was superior to other intralesional injection therapies(Table 1).

TABLE 1 Outcome of bovine SOD (orgoteine) therapy for Peyronie's DiseaseBartsch, 23 pts Excellent (≈100%) resolution of pain Europ. J.Rheumatol. 7/15 patients penile deviation Inflamm., 1981 disappearedDanner 39 pts. 75% pain improved Prog. Reprod. Biol. 78% decrease ofplaque size Med., 1983 53% deviation improved Gustafson, 22 pts. 18/22patients significantly improved Europ. J. Urol., 1981 Berlin 31 pts. 50%pain improved J. Urol., 1986 50% improved De Vries, 35 pts. 21/25patients significantly improved Akt. Urol.,1988 10 patients treatmentdiscontinued Schilling 78 pts. 72% pain improved Symposium 1985 33%decrease ofplaque size 33% deviation improved Pisani, 1985 70 pts. 90%pain improved 50% decrease of plaque size 50% deviation improved

However, serious allergic adverse reactions in individual patientsfollowing intraarticular injections of SOD led to disapprovement ofbovine SOD for treatments in humans.

The development of a procedure for bacterial production of recombinanthuman Cu/Zn SOD (rhSOD) reduced the risk of allergic side effects of SODtherapy. Although there is a minimal difference in the amino acidsequence of bovine SOD and rhSOD, the mechanism of action and efficacyof oxygen radical dismutation is equivalent.

In an embodiment according to the invention, for example, rhSODincorporated in liposomes is formulated as an ointment or gel, theoptionally admixed carrier being low-fat or fat-free and originatingfrom the group consisting of the organic and inorganic hydrogels. TheSOD compositions according to the invention, preferably rhSOD inliposomes, optionally mixed with at least one physiologically acceptablecarrier and/or hyaluronic acid, and optionally further additives, may bein the form of emulsions, suspensions, solutions, lotions or at leastlow-viscosity ointments or gels.

Another embodiment or feature of the invention relates to the use ofSOD, in particular of recombinant human SOD (rhSOD) in liposomes,optionally mixed with hyaluronic acid and/or a physiological acceptablecarrier and optionally further additives usually used for pharmaceuticalformulations, for the preparation of pharmaceutical compositions againstbums, scalds and radiation damage, in particular that caused by UVradiation.

The surprising success in external topical treatment of this type oftissue damage may be due to the fact that the tissue injury results inincreased formation of macrophages which, in the course of theirimmunological protective function (defense against infection,elimination of cell fragments), come into contact with the liposomes,break up their lipid layer and thus liberate the content—the SODmolecules—whereupon these in turn can display their superoxide-degradingand hence also tissue-protecting activity.

In an embodiment according to the invention, for example, rhSODincorporated in liposomes is formulated as a germ-free wound gel, theoptionally admixed carrier being low-fat or fat-free and originatingfrom the group consisting of the organic and inorganic hydrogels, and isapplied directly to the burn wound. In addition to clinical use forpatients with second and third degree burns, for example from accidents,such a formulation is also particularly useful for persons who havesuffered extensive or localized sunburns during sunbathing by water orin the snow.

Particularly in the case of burn wounds, it has also proven particularlyadvantageous if the SOD-containing liposomes are applied to the injuredareas in liquid form by spraying on from a spray can or spray bottle.This avoids direct contact of the wound with the fingers or another aidfor application, for example of a gel, and thus reduces the danger of anadditional infection.

In another embodiment, liposomally incorporated SOD, optionally withhyaluronic acid and/or further additives, is appliedto—preferably—sterile wound plaster and/or wound dressings, in orderthus to have a rapidly available and easily handled material for theeffective treatment and/or self-treatment of small and medium-sized burnwounds or skin burns at the place of occurrence. The particularadvantage of this application form is the simple manner of application,as it can also be carried out safely by medically untrained persons, forexample by parents whose child has burnt its finger on the cooker or hasscalded itself with hot water.

A particular aspect of the present invention is the use of SOD, inparticular rhSOD in liposomes, for the preparation of compositions whichare used before, during or after exposure to radiation. For purelytherapeutical applications, i.e., after excessive exposure to radiation,for example in the case of sunburn, it is advisable, in order to reducethe consequences of the exposure to radiation, to use the SODcompositions according to the invention, preferably rhSOD in liposomes,optionally mixed with at least one physiologically acceptable carrierand/or hyaluronic acid, and optionally further additives, in the form ofemulsions, suspensions, solutions, lotions or at least low-viscosityointments or gels.

Compositions which can be applied to the damaged skin areas by means ofan atomizing apparatus, for example a spray, are particularlyadvantageous. On the one hand, this avoids direct contact and hencepossible infection of the injured skin with possibly dirty fingers orother aids for application of the composition and, on the other hand, amore or less painful application by rubbing, for example of a relativelyhighly viscous gel, is thus dispensed with.

For the prophylactic and simultaneously therapeutic use before andduring exposure to radiation, particularly suitable compositionsaccording to the invention are those which, in addition to SOD andoptionally hyaluronic acid, also contain at least oneradiation-filtering or radiation-absorbing protective agent, preferablya light filter or UV absorber, in particular a UVB filter. Furthersubstances, especially skin care factors, may also be present.

As a result of the presence of SOD and optionally of hyaluronic acid inaddition to conventional light filter substances, the unpleasantconsequences of a sunburn can be considerably reduced. Such prophylacticand therapeutic sunscreen preparations can advantageously be used forany kind of sunbathing, whether on the beach, on the mountains, at sea,on the ski slope or in a solarium. In particular, people with sensitiveskin and those skin types which are not tanned by UV radiation benefitmost from this application form of the present invention.

Another embodiment relates to the preparation of compositions fortherapeutic and/or prophylactic use in the case of acute and chronicinflammations and inflammatory processes, rheumatic diseases, inparticular rheumatic joint inflammations and/or osteoarthritis, as wellas inflammations of the respiratory tract, in particular bronchitis,acute respiratory distress syndrome (ARDS), emphysema and otherinflammatory processes. Promising possibilities are thus opened upespecially for the area of cosmetic applications for prophylactic and/ortherapeutic treatment of local inflammations of the skin, such as, forexample, furuncles or acne, which are known to constitute a considerablecosmetic impairment for many people.

In one embodiment according to the invention, for example, rhSODincorporated in liposomes is formulated as sterile wound gel, theoptionally admixed carrier being low-fat or fat-free and originatingfrom the group consisting of the organic and inorganic hydrogels, and isapplied directly to the inflamed area. A low-fat or fat-free carrier isadvantageous in particular for open, inflamed wounds, since increasedlipid concentrations in the wound area may lead to the formation oftoxic degradation products. Hyaluronic acid can be particularlyadvantageously used in such compositions, either as the only carrier ormixed with at least one further carrier of the above category. Moreover,further suitable additives, as are usual, for example, forpharmaceutical formulations, may also be present in the compositionsaccording to the invention.

Particularly in the case of surface inflammations, it has also provenparticularly advantageous if the SOD-containing liposomes are applied tothe inflamed area by spraying on from a spray can, spray bottle or otherspray apparatus. This avoids direct contact of the wound with thefingers or with another aid for application of the composition and thusreduces the danger of an additional infection. In certain cases,however, it may also be advantageous if at least one of the compositionsaccording to the invention is applied on—optionally sterile—wounddressings, for example wound coverings, sticking plaster and the like.Wound dressings prepared in this manner are suitable for simple andrapid treatment, optionally also self-treatment, for example in the caseof inflamed wounds and/or rheumatic joint inflammations.

In a further embodiment of the present invention, liposomallyincorporated SOD, in particular rhSOD, together with hyaluronic acid andoptionally at least one additional low-fat or fat-free carrier, inparticular from the group consisting of the organic and inorganichydrogels, and optionally further additives, is formulated as aninjection solution and successfully used for rheumatological and/ororthopedic indications, for example rheumatic joint inflammations orosteoarthritis.

Injection may be given both directly into the affected joints(intraarticularly) or body regions or in another parenteral, preferablyintravenous, manner.

To a certain extent, rheumatic-arthritic symptoms can also be relievedby external treatment, for example by an external application, alsoaccording to the invention, of SOD in liposomes, in particular mixedwith hyaluronic acid. The active substances can be formulated and usedhere both in the form of ointments or gels, if necessary mixed withadditional, preferably low-fat or fat-free, carriers, in particular formthe group consisting of the organic and inorganic hydrogels, and in theform of sprays or tinctures. Suitable additives, as usually used forpharmaceutical formulations, may also be present in the compositions. Insome cases, an accompanying, oral administration of liposomal SOD in theform of tablets, capsules, sugar-coated tablets, powders, etc., may alsohave a supporting effect.

In a further embodiment, certain degenerative phenomena, such asemphysema, in particular cutaneous emphysema, could be successfullyreduced and suppressed by the use, according to the invention, of SOD inliposomes, optionally mixed with hyaluronic acid and/or at least onephysiologically acceptable carrier. Here too, additional,phagocytosis-stimulating effects of the SOD aggregates in the liposomesmay play a synergistic role, in addition to the purely enzymatic actionof the SOD.

The same is also likely to be the case for the successful therapeuticuse of the pharmaceutical compositions of the present invention in thecase of inflammations of the respiratory tract and of the lungs, suchas, for example, bronchitis, acute respiratory distress syndrome (ARDS)and pulmonary emphysema. In this case, particularly the application of asolution or emulsion of liposomally incorporated SOD by inhalation hasproven particularly suitable.

The use of the liposomal SOD composition is very successful particularlyin the area of cosmetic disorders. Small local inflammations, furuncles,acne and similar phenomena can be effectively treated by the use,according to the invention, of the SOD-containing compositions, inparticular in the form of ointments, gels or creams or in liquid form byspraying on. In the case of repeated, preferably regular, application, acertain prophylactic effect with respect to the formation of new skininflammation, for example of furuncles, can also be achieved. This toomay be associated with an activation of phagocytosis processes which istriggered or stimulated by the SOD aggregates.

With the use of a mixture of liposomal SOD and hyaluronic acid,moreover, the healing of the inflammation(s) is achieved with formationof new, more elastic and especially smoother tissue in comparison withhyaluronic acid-free compositions. This is a considerable advantageespecially for cosmetic applications on visible parts of the body, forexample on the face.

The compositions, according to the invention, of the present inventionare most effective when, depending on the use and method of theapplication, the SOD is present in a concentration of ≧0.01% by weight,in particular of 0.01 to 5% by weight, and the optionally additionallypresent hyaluronic acid in an amount of ≧0.05%, in particular 0.1 to 5%by weight, based on the prepared, ready-to-use composition. For topicalapplications, an amount of 0.01 to 1 mg SOD/cm² lesion area or bodysurface area has proven most suitable.

In particular for the treatment of induratio penis plastica, topicalapplications of from 0.01 to 1 g, preferably 0.1 to 1 g, most preferably0.5 g of an rhSOD gel/ointment containing the foregoing amounts ofactive ingredients is effective to penetrate into the underlying tissuesand treat/alleviate the symptoms of the disease. Preferably, thepharmaceutical composition is topically administered such that in asingle dose, 0.1 to 10 mg rhSOD is administered.

On the other hand, oral or parenteral applications are advantageouslycarried out with SOD doses of 0.5 to 50 mg/kg body weight, the doseadvantageously being adjusted to 0.5 to 10 mg/kg·day for repeated SODdoses during a therapy.

The present invention furthermore relates to the use of SOD for thepreparation of compositions which can be used for improving thestability of various organic, preferably biogenic, materials, and toprocesses for improving the stability of such organic materials by theuse of the compositions according to the invention.

In principle, SOD, preferably rhSOD in liposomes, in the form of anaqueous solution or emulsion with or without hyaluronic acid can beformulated according to the present invention and used directly for apreservative application. In various cases, however, the admixture ofone or more suitable carrier substances and/or further additives usuallyused for pharmaceutical compositions may be advantageous, for examplefor permitting or simplifying a certain, desired application form.

However, what is important for the use, according to the invention, ofthe compositions, for example in a process for improving the stabilityof the organic materials according to the present invention, is that theorganic material is brought at least partially into contact with atleast one of the compositions described here.

In an embodiment of the present invention, liposomally incorporated SOD,in particular rhSOD, is initially taken in a suitable buffer solution inorder to preserve organs or organic tissue which were removed from adonor for the purpose of transplantation, in the period between removaland reimplantation. A composition having an SOD content of 0.1-1% byweight has proven usefil here. The organ-or tissue preserving treatmentis best carried out by first irrigating the relevant organ by aconventional method of organ preservation, in particular by injection orinfusion of the liquid composition into the organ, for example a heart,a kidney, a liver, etc., and preferably then by immersing or preferablyplacing the respective organ or tissue part in a bath which alsocontains liposomal SOD either in the same buffer or in another suitablebuffer. This makes it possible on the one hand considerably to extendthe period of temporary storage and on the other hand to reduce possibleorgan or tissue damage after reimplantation.

Gratifyingly, it has been found that an additional synergistic effect ofthe type described above could be achieved in this case by mixinghyaluronic acid, for example in concentrations of about 0.05-2.5% byweight, with the existing emulsion of liposomal SOD and buffer.

In another embodiment, liposomal SOD in a liquid composition—with andwithout hyaluronic acid—is applied to foods, for example by means ofspray bottles, spray cans or other spray apparatuses. In particular,highly perishable meat products as well as dairy products of all kindsand fruit and vegetables could be decisively improved in their stabilityby means of these simple measures which can easily be applied. Theliposomally packaged SOD acts as a sort of antioxidant in this case.

Immersion baths which contain at least one of the compositions accordingto the invention and in which the organic materials, for example variousfoods, are immersed or placed over a desired period are also suitablefor bringing the active substances SOD and optionally hyaluronic acidinto contact with the organic material.

A further embodiment of the present invention relates to the use of freeSOD, in particular rhSOD, or liposomal SOD, mixed with hyaluronic acidand optionally at least one further suitable carrier, for improving thestability of cosmetic preparations based on organic substances, inparticular of skin care agents. Experiments have shown that both SOD inliposomes alone and free or liposomally incorporated SOD mixed withhyaluronic acid are very suitable for improving the stability of theorganic material in cosmetic preparations, such as ointments, creams,gels, lotions, waters, milks and the like when added to the latter.Particularly in the case of the mixture with hyaluronic acid, anadditional positive effect is obtained with regard to a certain increasein the suppleness and smoothing of the skin after application ofcosmetics improved in this manner.

The concentration of SOD in this application is preferably between 0.1%and 5% by weight, whereas the amount of optionally present hyaluronicacid is most preferably chosen as 0.5-5% by weight, based on the finalcomposition. If moreover one or more carrier substances are to beadmixed, they should preferably be low-fat or fat-free and optionallyoriginate from the group consisting of the organic and inorganichydrogels.

SOD concentrations of from 0.1 to 100 mg/kg of organic materials haveproven useful for improving the stability of organic materials, such as,for example, vegetable and animal tissues, organ transplants, foods, inparticular highly perishable meat and sausage products, or cosmeticsbased on organic substances.

In order further to explain the potential applications according to theinvention and the mode of action of the compositions described herein,some Examples are given below. The Examples serve for betterunderstanding of the present invention.

EXAMPLE 1

Use for Scalds

70 albino rabbits were divided into 7 groups (A-(G). An area of 5×10 cmon the back of each rabbit was shaved. After deep anesthetization, theshaved back area of each individual rabbit was dipped for exactly 16seconds into a tray having an opening of exactly 3×5 cm and filled withhot water at 93.5-95° C. This resulted in second to third degree scalds.The scalded areas were treated according to Table 1 below andimmediately covered with sterile wound dressing with an aluminum. inlay.The dressing was changed daily.

The liposomes with incorporated rhSOD were prepared as follows: theinjection method according to Batzri and Korn (Batzri, S. Kom, E. D.,1973, Biochim. Biophys. Acta 298:1015-19) was used as the preparationmethod for the largely multilaminar liposomes. The lipid componentsL-a-phosphatidylcholine, dipalmitoyl (DPPC) cholesterol and stearylamineare dissolved in 96% ethanol in a molar ratio of 7:1:2. The lipidconcentration of the liposome solution is 10 μmol/ml. The ethanol volumeis chosen so that the ethanol concentration in the preparation is lessthan 7%. The lyophilized rhSOD is dissolved in PBS. The proteinconcentration of the aqueous phase is 10 mg/ml. Both solutions arethermostated at 37° C. The liposomes are produced by continuousinjection of the ethanolic phase into the aqueous phase. To separate offthe unincorporated rhSOD or the ethanol, the liposome solution issubject to diafiltration against PBS (ultra/diafiltration unit fromAmicon; membrane; YM 100).

For the final formulation, the carbogel (Carbopol®, highly acidicacrylic acid polymers having a high molecular weight, DAB9) isrehydrated in distilled water and the pH is, adjusted to 7.5. Theliposome solution and the rehydrated gel are homogeneously mixed andstored at 4° C. The entire preparation process is carried out usingsterile solutions and in laminar flow conditions (sterile cleanroombench).

Preparation of the liposomally incorporated rhSOD-hyaluronic acid gel:Lyophilized hyaluronic acid is rehydrated in the aqueous liposomesolution, and the prepared gel is stored at 4° C.

The above-mentioned experimental animals were examined continuously by aveterinarian, and no signs of a clinical disease in the animals werefound during the course of the experiment. The animals also showed noposttraumatic pain symptoms after waking from the anaesthesia, butposttraumatic analgesia was initiated as a precaution.

Group F remained untreated as the control group. The remaining groupswere treated according to the following scheme:

TABLE 1 Test Number of Identification group animals numbers Dose andadministration A 10 1-10 0.1 mg of rhSOD/cm² of the lesion, inliposomes; applied 2 x on the first day B 10 11-20 1 mg of rhSOD/cm² ofthe lesion, in gel without liposomes; applied 1 x on the first day C 1021-30 1 mg of rhSOD/cm² of the lesion; intralesionally D 10 31-40Control group: gel with empty liposomes; applies 1 x on the first day E10 41-50 Control group: gel with liposomes; applied 1 x on the first dayF 10 51-80 Control group: no treatment G 10 61-70 0.1 mg of rhSOD inliposomes/cm² of the lesion, with hyaluronic acid; applied 2 x on thefirst day

The size of the wounds (planimetry, photographic documentation), thelocal status (color, presence of necroses, signs of epithelization,contractures, hair, cutimetry, granulation tissue), histopathologicalskin examinations and macroscopically, the general course of healing(photographic documentation) were evaluated statistically.

For the parameters investigated, a positive effect of the rhSODincorporated in liposomes on the healing regeneration processes in thetreated experimental animals was found. The effect of the rhSOD 24 hoursafter production of the lesion was most clear. Thus, for example, therewas a significant difference in lesion size between the test groups, the10 transition zone of the necrosis into the surrounding unscalded skinbeing substantially less affected by the scald in the rhSOD-treatedanimals. Histological examinations, too, confirmed the macroscopicresults. The most advantageous histopathological picture was found fortest groups A and G. Histologically, test groups A and G were the onlyones of all test groups to show no residual necroses after 4 weeks. Theoverall findings for groups A and G gave the following picture:

the lesion size was unchanged in comparison with the other test groupsafter 24 hours;

the width of the oedema. on the other hand was substantially reduced andaggression was accelerated;

there were no residual necroses in the corium;

test group G moreover exhibited the formation of smoother and moreelastic new tissue compared with all other test groups.

In an observation period of 24 hours, the positive effect of the rhSODincorporated in liposomes could be clearly demonstrated although thedose of the rhSOD in test groups A and G was ten times lower than intest groups B and C. The after-burning phenomena of the scald wounds andin particular the oedema declined fastest and most substantially in thegroups treated with liposomally incorporated rhSOD.

EXAMPLE 2

Use for Burns

An experiment with the production of bum wounds was carried outanalogously to the method of M. Choi and H. G. Ehrlich (American Journalof Pathology 142, 1993, 519-529). 40 rats (Wistar, male rats) of 300-350g body weight were kept under standard conditions. Bum wounds in theform of a strip pattern were made on the anaesthetized rats (anesthetic:pentobarbital, i.p.) with a stamp which was heated in boiling water. Forthis purpose, the stamp was pressed for 30 seconds against the shavedskin of the animals. In order to obtain the desired pattern on the skin,the stamp according to Choi and Ehrlich (1993) had a comb-like design.

The rats were divided into 4 groups of 10 animals each (Table 2).

TABLE 2 Number of Test group animals Administration 1 10 Control group:no treatment 2 10 Carrier gel with empty liposomes 3 10 Gel with rhSODin liposomes 4 10 rhSOD in liposomes with hyaluronic acid in carrier gel

The preparation of the liposomes and of the gels was carried outanalogously to Example 1. The rats were treated six times at regularintervals in the course of 48 hours, the first treatment being carriedout a few minutes after production of the lesion. Liposomally packagedrhSOD was used in an amount of 0.05 mg/cm² of the lesion, and the amountof hyaluronic acid in the composition used was 3% by weight.

The planimetry and the macromorphology of the wound pattern which isbrought about by the design of the stamp were used as an evaluationcriterion for the potential action. Furthermore, standard histology(haematoxylin, eosin and vital staining; according to H. Millesi, 1970,Chirurgia Plastica et Reconstructia Vol. 8, Springer Verlag Berlin,Heidelberg, New York) of the t issue samples was carried out. Sampleswere taken after 0 h, 25 h, 72 h, 7 days and 21 days.

In groups 3 and 4, the pattern produced by the stamp shape was retained,i.e., the burned strips 10 remained vital. In groups 1 and 2, on theother hand, confluence to a more or less uniform wound surface occurred.In groups 3 and 4, an advantageous histopathological picture was alsoobserved. In the groups treated with rhSOD incorporated in liposomes,the pattern of the unexposed strips was maintained. The surfacesaffected by the stamp pressure were replaced by granulation and scartissue, the majority of which was necrosis-free. In groups 1 and 2, auniform scar surface without a pattern and with some 20 deep necroseswas observable.

EXAMPLE 3

Use for Radiation Damage

Hairless mice were exposed to the minimum erythema dose (MED) of UVradiation. The experimental 25 setup was as described by B. C. Pence andM. F. Naylor (B. C. Pence, M. F. Naylor 1993, J. of InvestigativeDermatology 95: 213-16). A single dose of UVB radiation (290-320 nm)from a Westinghouse FS-40 sun lamp was used in such a way that the micewere exposed to a total energy of 0.09 J/cm². This dose corresponds tothree times the MED for Caucasian volunteers. 40 mice in 4 groups of 10animals each were used (Table 3).

TABLE 3 Number of Test group animals Administration 1 10 Control group:no treatrnent 2 10 Carrier gel with empty liposomes 3 10 Gel with rhSODin liposomes 4 10 rhSOD in liposomes with hyaluronic acid in carrier gel

The liposomes and gels were prepared analogously to Example 1. The gelwas applied immediately (2-3 min) after exposure to radiation and thentwice more at regular intervals of 4 hours. The gel in the compositionused contained 0.5% by weight of hyaluronic acid, and the administereddose of rhSOD was 0.5 mg/cm² of irradiated body surface. A visual colorcomparison of all animals at regular time intervals was carried out asan evaluation criterion. Furthermore, the occurrence of “sunburn cells”was tested.

In groups 3 and 4, a significantly lower erythema in comparison withgroups 1 and 2 occurred after 10 hours. After 24 hours, tissue sampleswere taken and were tested for the characteristic “sunburn cells”. Ingroups 3 and 4, no “sunburn cells” or only isolated “sunburn cells”occurred. The intensity of redness could be correlated with theoccurrence of sunburn cells.

EXAMPLE 4

Comparison of Different Application Forms

140 male OF1 (outbred) mice (from the Institute for Experimental AnimalBreeding and Keeping, Himberg, Austria), all between 8 and 10 weeks old,were divided into 14 groups (A-N) according to the randomizationprinciple. Under ether anaesthesia, the mice were inoculatedintranasally with 50 μl of PBS solution which contained 1×10⁵ PFU/mouse(=2-2.5×LD₅₀) of the influenza virus A/WSN/33 (referred to below asWSN). The treatment of the WSN-infected mice began on the 4th day afterinfection, i.e., at the beginning of the occurrence of clinical symptomsof the influenza disease in the mice. The mice were treated once dailyup to day 11 after the infection. They were treated topically,subcutaneously, intravenously and intranasafly with preparations whichcontained rhSOD in the following liposomal vesicle types:

a) rhSOD in small, unilamellar vesicles (SUV), size≦200 nm,preferably≦about 100 nm,

b) rhSOD in injection vesicles (IV), diameter≦about 200 nm, and

c) rhSOD in multilamellar vesicles (MLV), diameter≦about 500 nm.

Table 4 shows the experimental setup and the results obtained. For thetopical treatment, a liposomal rhSOD gel was applied to the ventral sideof the mice, especially in the breast region, by rubbing in once perday. A skin area of about 10 cm² was thus covered with an approximately2 mm thick gel layer. The concentrations stated in Table 4 relate to thetreated area in cm². The intranasal application of rhSOD took place withthe animals under ether anesthesia. 0.05 ml of a suspension containingrhSOD in IV (1 mg/ml) was introduced into the nostrils of the mice bymeans of a micropipette. An injection was given into the caudal vein forintravenous administration of the rhSOD suspensions (cf. Table 4), whilethe same suspensions as for the i.v. injection were injectedsubcutaneously into the neck and/or shoulder region for the subcutaneousapplication. The number of deaths among the mice did not increase afterthe 15th day after the infection (period tested: day 15 to day 25 afterthe infection), so that mice which have survived on the 15th day wereregarded as having been cured of the influenza infection.

The pathogenesis of an influenza virus infection in mice tends toindicate a hyperreaction of the immune defense of the host organism as adirect effect in relation to the multiplication of the virus. Theformation of free oxygen radicals in the course of an influenzainfection is influenced by the massive infiltration of lymphoid cellsinto the lung tissue and by increased xanthine oxidase activity in thelungs and in the serum of the mice (Oda et al., Science, 1989, 244(4907): 974-6).

Survivors per 10 Group Application form and dose mice A Topicalapplication of a gel of rhSOD incorporated in 4 out of 10 SUV; dose:0.15 mg rhSOD/cm² treated surface B Topical application of a gel ofrhSOD incorporated in 4 out of 10 IV; dose: 0.15 mg rhSOD/cm² treatedsurface C Topical application of a gel of rhSOD incorporated in 3 out of10 MLV; dose: 0.42 mg rhSOD/cm² treated surface D Topical application ofa gel with IV without rhSOD 2 out of 10 E s.c. application of asuspension of rhSOD incorporated 8 out of 10 in SUV; dose: 0.5 ml;suspension: 1 mg rhSOD/ml F s.c. application of a suspension of rhSODincorporated 8 out of 10 in IV; dose: 0.5 ml; suspension: 1 mg rhSOD/mlG s.c. application of a suspension of rhSOD incorporated 5 out of 10 inMLV; dose: 0.5 ml; suspension: 3 mg rhSOD/ml H s.c. application of 0.5ml of an IV suspension without 1 out of 10 rhSOD I i.v. application of asuspension of rhSOD incorporated 9 out of 10 in SUV; dose: 0.5 ml;suspension: 1 mg rhSOD/ml J i.v. application of a suspension of rhSODincorporated 10 out of 10  in SUV; dose: 0.5 ml; suspension: 1 mgrhSOD/ml K application of a suspension of rhSOD incorporated in 4 out of10 MLV; dose: 0.5 ml; suspension: 3 mg rhSOD/ml L i.v. application of asuspension of rhSOD incorporated 1 out of 10 in SUV; dose: 0.5 ml;suspension: 1 mg rhSOD/ml M i.n. application of a suspension of rhSODincorporated 8 out of 10 in IV; dose: 0.05 ml; suspension: 1 mg rhSOD/mrN Control group without treatment 1 out of 10

Result

Mice which were treated parenterally (s.c., i.v. and i.n.) with rhSOD inSUV or IV liposomes survived in most cases (groups E, F, L, J, M) incomparison with the control groups which were either not treated at allor treated only with liposomes without SOD (groups H, L and N). It wasalso found, surprisingly, that mice which were treated with rhSOD in MLVwere less well protected against the fatal influenza infection althoughin these liposomes preparations a larger amount of rhSOD was used(groups C, G and K). The reason for this is probably the vesicle size(in the region of 500 nm; for comparison: IV≦5 about 200 nm; SUV≦about100 nm), poorer distribution and a lower degree of fusibility of the MLVwith the cells, as was established on the basis of an in vitro fusionassay (fusion assay: CHO cells were incubated overnight with 20 μmol ofrhSOD-containing IV and MLV liposomes, and rhsbD was detected byimmunofluorescence after treatment with 1% Triton X100).

The topical treatment of the influenza-infected mice with rhSOD gelresulted in only partial protection against the fatal consequences ofthe disease; nevertheless, this protection was significant in the groupswhich were treated with SUV or IV preparations (group A and B. Theresults show that free oxygen radicals play an important role withregard to the fatal effects of the influenza infection in mice and thatrhSOD, incorporated in small liposomal vesicles (SUV and IV), has aconsiderable therapeutic potential with respect to this viral infection.

The results also show that rhSOD, incorporated in small liposomes (SUV,IV) having a diameter of about 200 nm or less, evidently can overcomethe skin barriers even in the case of essentially healthy, intact skinand can penetrate into relatively deep tissue layers in order to displayits protective action against the viral infection in the lung region(pneumonia). Liposomally incorporated rhSOD can therefore be used notonly for i.v., s.c. and i.n. applications but also in the form ofointments, gels, creams or other suitable formulations, optionally incombination with other active substances and/or including furtheradditives, as an effective therapeutic agent for the treatment ofdiseases which are associated with free oxygen radicals, for exampleof—optionally microbial—inflammations, in particular of the upperrespiratory tract and of the lung.

EXAMPLE 5

Topical Application in the Case of Herpes Labialis

In a human experiment, rhSOD, incorporated in small, unilamellarvesicles a (SUV) having a vesicle diameter of about 100 nm or less, wastested against a local skin inflammation in the mouth area caused byfever blisters on the lip (herpes labialis). Herpes infections andherpes diseases are known to be difficult to treat and in general veryunpleasant for the persons affected, that is to say frequently causepain (e.g. in the case of herpes zoster), itching, burning, weepingand/or cosmetically disadvantageous effects, especially in the facearea.

The liposomal rhSOD preparation based on Carbopol® gel had a consistencysimilar to that of a skin cream and was applied in a concentration ofabout 0.15 mg of rhSOD per cm² of treated area, once or twice per day,directly to the inflamed area. Noticeable relief was observed within afew hours after only the first application, and the typical symptoms hadvery largely died away after treatment for only 3 days.

EXAMPLE 6

Topical application against allergy

The same rhSOD preparation as in Example 5 was tested in another humanexperiment against allergic reactions. The allergy symptoms were redeyes and swellings around the eyes and in the region of the eyes andnose, possibly as additional effects of an existing hay fever allergy.In addition to the unpleasant facial tensions caused by the swelling andthe psychological suffering additionally associated with this,impairment of normal vision was also present.

The rhSOD gel was applied, analogously to Example 5, in a concentrationof about 0.15 mg of rhSOD per cm² of treated area in the entire affectedfacial area, in the same way as a skin cream. The result was impressive:after only a single application, the swellings disappeared almostcompletely; as a precaution, cream was applied a second time on thefollowing day, but a further treatment was no longer necessary. Thesymptoms had virtually completely disappeared.

EXAMPLE 7

Use against psoriasis

In a further experiment, the rhSOD gel from Example 5 was used to treatpsoriasis in a child. The preparation was applied twice a day (in themorning and in the evening), analogously to Example 5, in aconcentration of about 0.15 mg of rhSOD per cm² of treated area, to allaffected parts of the body, in the same way as the skin cream.

Result

After treatment for three days, a substantial decline in the reddeningof the skin in the region of the inflammation center and a reduction initching were observed. This gratifying finding shows that, with externaltopical application, rhSOD in liposomes can also be used successfully inpsoriasis patients, at least to relieve the symptoms—even if not to curethe disease.

EXAMPLE 8

Use against IPP (Peyronie's Disease)

In a further experiment, 20 patients (31 to 67 years old) withPeyronie's disease were treated with a gel preparation containing 1.5 mgliposomal rh-SOD/g (gel basis: Carbopol® powder neutralized andcross-linked by addition of NaOH; pH 7; see also Example 5) in aprospective, uncontrolled study. The duration of disease at thebeginning of the study was less than one year in 15 patients and betweenone and five years in 5 patients. Four patients had undergone priortherapies: three of them had been treated by iontophoresis ofdexamethasone and verapamil, and one patient had been operated about oneyear before rh-SOD therapy (Nesbit procedure).

Patients judged pain according to a pain scale. The plaques weremeasured with a measuring tape, or with ultrasound if not easilypalpable. An x-ray examination was performed to exclude calcifications.Penile deviation was photodocumented in two planes. If erectiledysfunction was present, intra-cavernous prostaglandine E1 was injectedfor diagnostic evaluation and the grade of deviation judged by theinvestigator. In addition, any impairment of sexual activity caused byPeyronie's disease was recorded.

Documentation of skin reactions was planned according to the Lent SomaScore. All possible adverse reactions were recorded, and patients wereasked to complete a questionnaire concerning the efficacy andconvenience of the treatment as well as their quality of life.

Patients were instructed to store the liposomal rhSOD preparation in therefrigerator and to administer approximately 0.5 g of the preparationtwice daily. Controls were performed on day 3 (to exclude adversereactions), day 10, day 21 and day 42 after onset of therapy. SODtherapy was continued to a maximum of 6 weeks and only terminatedearlier on patient's request.

Results

Pain relief was reported by 13 of 13 patients: complete elimination ofpain occurred in 7 of 13 patients, in 2 patients as early as 3 daysafter the onset of therapy, whereas 6 of 13 patients observed an almostcomplete (>90%) resolution of pain. SOD therapy also reduced pain at thesite of prior surgical repair in one patient. A measurable reduction ofplaque size was observed in 8 of 14 patients, whereas minimalimprovement of deviation was demonstrable in only 3 of 10 patients.Improvement of sexual function was reported by 12 of 15 patients, mainlydue to a reduction of pain during erections (in 11 patients).

Deterioration of penile deviation and an increase of plaque size despiteimprovement of pain status was observed in one patient, who underwentsurgical correction of the penile curvature. Recurrence of Peyronie'ssymptoms occurred in another patient about 6 months after SOD therapyhad completely resolved pain and diminished plaque size. The patientrequested to undergo iontophoresis therapy.

SOD therapy was discontinued with 7 patients after 3 weeks: in 5patients because the symptoms had significantly improved, in 2 patientsbecause they had not noticed any change. The remaining 13 patientscontinued treatment over the whole 6 weeks period.

Patients judged SOD therapy very effective or effective in 85% and veryconvenient in 92%. No local side effects or systemic adverse reactionswere observed by either the investigators or the patients. Thequestionnaires revealed a significant impact of Peyronie's disease onthe quality of life in 7 patients. Following SOD therapy, 5 patientsreported a significant improvement of quality of life, whereas the 2patients with deterioration and recurrence of Peyronie's diseasereported no change.

Conclusion

Thus, topical application of liposomal rhSOD is a convenient treatmentwithout side effects that shows excellent results in initial Peyronie'sDisease symptoms (pain, plaque).

What is claimed is:
 1. A method for the prophylactic or therapeuticintervention of a pathological condition affecting a human body or partthereof, wherein said condition is susceptible to adminstration ofsuperoxide dismutase, the method comprising topically administering tothe human body or part thereof a pharmaceutical composition comprising alow-fat or fat free carrier together with recombinant human Cu,Zn-SOD(rhSOD) in unilamellar liposomes having an average diameter of 200nm or less in an amount effective for penetrating from the outside intodeeper lying tissue layers of the body or part thereof, to prevent orrelieve said pathological condition, and wherein said pathologicalcondition is induratio penis plastica (IPP).
 2. A method according toclaim 1, wherein the pharmaceutical composition further compriseshyaluronic acid.
 3. A method according to claim 2, wherein thehyaluronic acid is present in a concentration of from 0.05 to 5% byweight based on the pharmaceutical composition.
 4. A method according toclaim 1, wherein the pharmaceutical composition is administered in theform of emulsions, suspensions, solutions, lotions, ointments or gels,or by spraying from a spray apparatus.
 5. A method according to claim 1,wherein the pharmaceutical composition is administered in a dose of0.1-10 mg rhSOD per single administration.
 6. A method according toclaim 5, wherein the pharmaceutical composition is administered twicedaily.
 7. A method according to claim 1, wherein the rhSOD is present inthe pharmaceutical composition in a concentration of 0.01 to 5% byweight based on the pharmaceutical composition.
 8. A method according toclaim 1, wherein the low-fat or fat free carrier is selected from thegroup consisting of organic and inorganic hydrogels.